Consensus Paper: Cerebellum and Reward.

Cerebellum is a key-structure for the modulation of motor, cognitive, social and affective functions, contributing to automatic behaviours through interactions with the cerebral cortex, basal ganglia and spinal cord. The predictive mechanisms used by the cerebellum cover not only sensorimotor functions but also reward-related tasks. Cerebellar circuits appear to encode temporal difference error and reward prediction error. From a chemical standpoint, cerebellar catecholamines modulate the rate of cerebellar-based cognitive learning, and mediate cerebellar contributions during complex behaviours. Reward processing and its associated emotions are tuned by the cerebellum which operates as a controller of adaptive homeostatic processes based on interoceptive and exteroceptive inputs. Lobules VI-VII/areas of the vermis are candidate regions for the cortico-subcortical signaling pathways associated with loss aversion and reward sensitivity, together with other nodes of the limbic circuitry. There is growing evidence that the cerebellum works as a hub of regional dysconnectivity across all mood states and that mental disorders involve the cerebellar circuitry, including mood and addiction disorders, and impaired eating behaviors where the cerebellum might be involved in longer time scales of prediction as compared to motor operations. Cerebellar patients exhibit aberrant social behaviour, showing aberrant impulsivity/compulsivity. The cerebellum is a master-piece of reward mechanisms, together with the striatum, ventral tegmental area (VTA) and prefrontal cortex (PFC). Critically, studies on reward processing reinforce our view that a fundamental role of the cerebellum is to construct internal models, perform predictions on the impact of future behaviour and compare what is predicted and what actually occurs.

Zona incerta modulation of the inferior olive and the pontine nuclei.

The zona incerta (ZI) is a subthalamic structure that has been implicated in locomotion, fear, and anxiety. Recently interest has grown in its therapeutic efficacy in deep brain stimulation in movement disorders. This efficacy might be due to the ZI's functional projections to the other brain regions. Notwithstanding some evidence of anatomical connections between the ZI and the inferior olive (IO) and the pontine nuclei (PN), how the ZI modulates the neuronal activity in these regions remains to be determined. We first tested this by monitoring responses of single neurons in the PN and IO to optogenetic activation of channelrhodopsin-expressing ZI axons in wild-type mice, using an in vivo awake preparation. Stimulation of short, single pulses and trains of stimuli at 20 Hz elicited rapid responses in the majority of recorded cells in the PN and IO. Furthermore, the excitatory response of PN neurons scaled with the strength of ZI activation. Next, we used in vitro electrophysiology to study synaptic transmission at ZI-IO synapses. Optogenetic activation of ZI axons evoked a strong excitatory postsynaptic response in IO neurons, which remained robust with repeated stimulation at 20 Hz. Overall, our results demonstrate a functional connection within ZI-PN and ZI-IO pathways.

The cerebellum tells the amygdala, "Nothing diminishes anxiety faster than action".

In this issue of Neuron, Zhang et al. question the neural substrates of exercise-based alleviation of anxiety in rodents. In brief, they propose a model where physical activity provides an anxiolytic effect by recruiting specific cerebello-limbic circuits.

The cerebellum directly modulates the substantia nigra dopaminergic activity.

Evidence of direct reciprocal connections between the cerebellum and basal ganglia has challenged the long-held notion that these structures function independently. While anatomical studies have suggested the presence of cerebellar projections to the substantia nigra pars compacta (SNc), the nature and function of these connections (Cb-SNc) is unknown. Here we show, in mice, that Cb-SNc projections form monosynaptic glutamatergic synapses with dopaminergic and non-dopaminergic neurons in the SNc. Optogenetic activation of Cb-SNc axons in the SNc is associated with increased SNc activity, elevated striatal dopamine levels and increased locomotion. During behavior, Cb-SNc projections are bilaterally activated before ambulation and unilateral lever manipulation. Cb-SNc projections show prominent activation for water reward and higher activation for sweet water, suggesting that the pathway also encodes reward value. Thus, the cerebellum directly, rapidly and effectively modulates basal ganglia dopamine levels and conveys information related to movement initiation, vigor and reward processing.

Elevated granulocyte colony stimulating factor (CSF) causes cerebellar deficits and anxiety in a model of CSF-1 receptor related leukodystrophy.

Colony stimulating factor (CSF) receptor-1 (CSF-1R)-related leukoencephalopathy (CRL) is an adult-onset, demyelinating and neurodegenerative disease caused by autosomal dominant mutations in CSF1R, modeled by the Csf1r+/- mouse. The expression of Csf2, encoding granulocyte-macrophage CSF (GM-CSF) and of Csf3, encoding granulocyte CSF (G-CSF), are elevated in both mouse and human CRL brains. While monoallelic targeting of Csf2 has been shown to attenuate many behavioral and histological deficits of Csf1r+/- mice, including cognitive dysfunction and demyelination, the contribution of Csf3 has not been explored. In the present study, we investigate the behavioral, electrophysiological and histopathological phenotypes of Csf1r+/- mice following monoallelic targeting of Csf3. We show that Csf3 heterozygosity normalized the Csf3 levels in Csf1r+/- mouse brains and ameliorated anxiety-like behavior, motor coordination and social interaction deficits, but not the cognitive impairment of Csf1r+/- mice. Csf3 heterozygosity failed to prevent callosal demyelination. However, consistent with its effects on behavior, Csf3 heterozygosity normalized microglial morphology in the cerebellum and in the ventral, but not in the dorsal hippocampus. Csf1r+/- mice exhibited altered firing activity in the deep cerebellar nuclei (DCN) associated with increased engulfment of glutamatergic synapses by DCN microglia and increased deposition of the complement factor C1q on glutamatergic synapses. These phenotypes were significantly ameliorated by monoallelic deletion of Csf3. Our current and earlier findings indicate that G-CSF and GM-CSF play largely non-overlapping roles in CRL-like disease development in Csf1r+/- mice.

Cerebellar Contributions to the Basal Ganglia Influence Motor Coordination, Reward Processing, and Movement Vigor.

Both the cerebellum and the basal ganglia are known for their roles in motor control and motivated behavior. These two systems have been classically considered as independent structures that coordinate their contributions to behavior via separate cortico-thalamic loops. However, recent evidence demonstrates the presence of a rich set of direct connections between these two regions. Although there is strong evidence for connections in both directions, for brevity we limit our discussion to the better-characterized connections from the cerebellum to the basal ganglia. We review two sets of such connections: disynaptic projections through the thalamus and direct monosynaptic projections to the midbrain dopaminergic nuclei, the VTA and the SNc. In each case, we review the evidence for these pathways from anatomic tracing and physiological recordings, and discuss their potential functional roles. We present evidence that the disynaptic pathway through the thalamus is involved in motor coordination, and that its dysfunction contributes to motor deficits, such as dystonia. We then discuss how cerebellar projections to the VTA and SNc influence dopamine release in the respective targets of these nuclei: the NAc and the dorsal striatum. We argue that the cerebellar projections to the VTA may play a role in reward-based learning and therefore contribute to addictive behavior, whereas the projection to the SNc may contribute to movement vigor. Finally, we speculate how these projections may explain many of the observations that indicate a role for the cerebellum in mental disorders, such as schizophrenia.

Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator.

Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.

Cerebellar neuronal dysfunction accompanies early motor symptoms in spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is an adult-onset, progressive ataxia. SCA3 presents with ataxia before any gross neuropathology. A feature of many cerebellar ataxias is aberrant cerebellar output that contributes to motor dysfunction. We examined whether abnormal cerebellar output was present in the CMVMJD135 SCA3 mouse model and, if so, whether it correlated with the disease onset and progression. In vivo recordings showed that the activity of deep cerebellar nuclei neurons, the main output of the cerebellum, was altered. The aberrant activity correlated with the onset of ataxia. However, although the severity of ataxia increased with age, the severity of the aberrant cerebellar output was not progressive. The abnormal cerebellar output, however, was accompanied by non-progressive abnormal activity of their upstream synaptic inputs, the Purkinje cells. In vitro recordings indicated that alterations in intrinsic Purkinje cell pacemaking and in their synaptic inputs contributed to abnormal Purkinje cell activity. These findings implicate abnormal cerebellar physiology as an early, consistent contributor to pathophysiology in SCA3, and suggest that the aberrant cerebellar output could be an appropriate therapeutic target in SCA3.

Potential Variables for Improved Reproducibility of Neuronal Cell Grafts at Stroke Sites.

Interest is growing in using cell replacements to repair the damage caused by an ischemic stroke. Yet, the usefulness of cell transplants can be limited by the variability observed in their successful engraftment. For example, we recently showed that, although the inclusion of donor-derived vascular cells was necessary for the formation of large grafts (up to 15 mm3) at stroke sites in mice, the size of the grafts overall remained highly variable. Such variability can be due to differences in the cells used for transplantation or the host environment. Here, as possible factors affecting engraftment, we test host sex, host age, the extent of ischemic damage, time of transplant after ischemia, minor differences in donor cell maturity, and cell viability at the time of transplantation. We find that graft size at stroke sites correlates with the size of ischemic damage, host sex (females having graft sizes that correlate with damage), donor cell maturity, and host age, but not with the time of transplant after stroke. A general linear model revealed that graft size is best predicted by stroke severity combined with donor cell maturity. These findings can serve as a guide to improving the reproducibility of cell-based repair therapies.

Subthalamic nucleus modulation of the pontine nuclei and its targeting of the cerebellar cortex.

The subthalamic nucleus (STN) has been implicated in motor and non-motor tasks, and is an effective target of deep brain stimulation (DBS) for the treatment of Parkinson's disease, likely in part due to the STN's projections outside of the basal ganglia to other brain regions. While there is some evidence of a disynaptic connection between the STN and the cerebellum via the pontine nuclei (PN), how the STN modulates the activity of the neurons in the PN remains unknown. Here we addressed this question using a combination of anatomical tracings, optogenetics, and in vivo electrophysiology in both wild-type and transgenic mice of both sexes. Approximately half of recorded neurons in the PN, which were located primarily in the medial area, responded with short latency to both single pulses and trains of optogenetic stimulation of channelrhodopsin (ChR2)-expressing STN axons in awake, head-restrained mice. Furthermore, the increase in the activity of PN neurons correlated with the strength of activation of STN axons, suggesting that the STN projections to the PN could, in principle, encode information in a graded manner. In addition, transsynaptic retrograde tracing confirmed that the STN sends disynaptic projections to the cerebellar cortex. These results suggest that the STN sends robust functional projections to the PN, which then propagate to the cerebellum, and have important implications for understanding motor control of normal conditions, and Parkinsonian symptoms, where this pathway may have a role in the therapeutic efficacy of STN DBS.Significance StatementThe primary excitatory nucleus in the basal ganglia, the subthalamic nucleus, is known to play a role in pathways modulating movement. The pontine nuclei are the main precerebellar nuclei, which transmit signals through their axonal projections to the cerebellum as mossy fibers. The pathway we have functionally characterized in this paper represents an additional cortex-independent pathway capable of relaying information between the basal ganglia and cerebellum. The effectiveness of subthalamic nucleus deep brain stimulation (STN-DBS) in Parkinson's disease suggests that this pathway could be explored as an avenue of investigation for therapeutic purposes.

Mechanism of stress-induced attacks in an episodic neurologic disorder.

Stress is the most common trigger among episodic neurologic disorders. In episodic ataxia type 2 (EA2), physical or emotional stress causes episodes of severe motor dysfunction that manifest as ataxia and dystonia. We used the tottering (tg/tg) mouse, a faithful animal model of EA2, to dissect the mechanisms underlying stress-induced motor attacks. We find that in response to acute stress, activation of α1-adrenergic receptors (α1-Rs) on Purkinje cells by norepinephrine leads to their erratic firing and consequently motor attacks. We show that norepinephrine induces erratic firing of Purkinje cells by disrupting their spontaneous intrinsic pacemaking via a casein kinase 2 (CK2)-dependent signaling pathway, which likely reduces the activity of calcium-dependent potassium channels. Moreover, we report that disruption of this signaling cascade at a number of nodes prevents stress-induced attacks in the tottering mouse. Together, our results suggest that norepinephrine and CK2 are required for the initiation of stress-induced attacks in EA2 and provide previously unidentified targets for therapeutic intervention.

Donor-derived vasculature is required to support neocortical cell grafts after stroke.

Neural precursor cells (NPCs) transplanted into the adult neocortex generate neurons that synaptically integrate with host neurons, supporting the possibility of achieving functional tissue repair. However, poor survival and functional neuronal recovery of transplanted NPCs greatly limits engraftment. Here, we test the hypothesis that combining blood vessel-forming vascular cells with neuronal precursors improves engraftment. By transplanting mixed embryonic neocortical cells into adult mice with neocortical strokes, we show that transplant-derived neurons synapse with appropriate targets while donor vascular cells form vessels that fuse with the host vasculature to perfuse blood within the graft. Although all grafts became vascularized, larger grafts had greater contributions of donor-derived vessels that increased as a function of their distance from the host-graft border. Moreover, excluding vascular cells from the donor cell population strictly limited graft size. Thus, inclusion of vessel-forming vascular cells with NPCs is required for more efficient engraftment and ultimately for tissue repair.

Chromatin architecture at susceptible gene loci in cerebellar Purkinje cells characterizes DNA damage-induced neurodegeneration.

The pathogenesis of inherited genome instability neurodegenerative syndromes remains largely unknown. Here, we report new disease-relevant murine models of genome instability­driven neurodegeneration involving disabled ATM and APTX that develop debilitating ataxia. We show that neurodegeneration and ataxia result from transcriptional interference in the cerebellum via aberrant messenger RNA splicing. Unexpectedly, these splicing defects were restricted to only Purkinje cells, disrupting the expression of critical homeostatic regulators including ITPR1, GRID2, and CA8. Abundant genotoxic R loops were also found at these Purkinje cell gene loci, further exacerbating DNA damage and transcriptional disruption. Using ATAC-seq to profile global chromatin accessibility in the cerebellum, we found a notably unique chromatin conformation specifically in Purkinje chromatin at the affected gene loci, thereby promoting susceptibility to DNA damage. These data reveal the pathogenic basis of DNA damage in the nervous system and suggest chromatin conformation as a feature in directing genome instability­associated neuropathology.

Navigating clues to success in academia.

Academic success and how to achieve it takes diverse forms, depending on who's asked. We suggest that happiness, impact, and longevity can be achieved with professional effort and support that balances the toil and joys of one's chosen path.

Acute cerebellar knockdown of Sgce reproduces salient features of myoclonus-dystonia (DYT11) in mice.

Myoclonus dystonia (DYT11) is a movement disorder caused by loss-of-function mutations in SGCE and characterized by involuntary jerking and dystonia that frequently improve after drinking alcohol. Existing transgenic mouse models of DYT11 exhibit only mild motor symptoms, possibly due to rodent-specific developmental compensation mechanisms, which have limited the study of neural mechanisms underlying DYT11. To circumvent potential compensation, we used short hairpin RNA (shRNA) to acutely knock down Sgce in the adult mouse and found that this approach produced dystonia and repetitive, myoclonic-like, jerking movements in mice that improved after administration of ethanol. Acute knockdown of Sgce in the cerebellum, but not the basal ganglia, produced motor symptoms, likely due to aberrant cerebellar activity. The acute knockdown model described here reproduces the salient features of DYT11 and provides a platform to study the mechanisms underlying symptoms of the disorder, and to explore potential therapeutic options.

Cerebellar modulation of the reward circuitry and social behavior.

The cerebellum has been implicated in a number of nonmotor mental disorders such as autism spectrum disorder, schizophrenia, and addiction. However, its contribution to these disorders is not well understood. In mice, we found that the cerebellum sends direct excitatory projections to the ventral tegmental area (VTA), one of the brain regions that processes and encodes reward. Optogenetic activation of the cerebello-VTA projections was rewarding and, in a three-chamber social task, these projections were more active when the animal explored the social chamber. Intriguingly, activity in the cerebello-VTA pathway was required for the mice to show social preference in this task. Our data delineate a major, previously unappreciated role for the cerebellum in controlling the reward circuitry and social behavior.

Increased susceptibility to cortical spreading depression and epileptiform activity in a mouse model for FHM2.

Migraine is a highly prevalent, debilitating, episodic headache disorder affecting roughly 15% of the population. Familial hemiplegic migraine type 2 (FHM2) is a rare subtype of migraine caused by mutations in the ATP1A2 gene, encoding the α2 isoform of the Na+/K+-ATPase, predominantly expressed in astrocytes. Differential comorbidities such as epilepsy and psychiatric disorders manifest in patients. Using a mouse model harboring the G301R disease-mutation in the α2 isoform, we set to unravel whether α2+/G301R mice show an increased susceptibility for epilepsy and cortical spreading depression (CSD). We performed in vivo experiments involving cortical application of KCl in awake head-restrained male and female mice of different age groups (adult and aged). Interestingly, α2+/G301R mice indeed showed an increased susceptibility to both CSD and epileptiform activity, closely replicating symptoms in FHM2 patients harboring the G301R and other FHM2-causing mutations. Additionally, this epileptiform activity was superimposed on CSDs. The age-related alteration towards CSD indicates the influence of female sex hormones on migraine pathophysiology. Therefore, the FHM2, α2+/G301R mouse model can be utilized to broaden our understanding of generalized epilepsy and comorbidity hereof in migraine, and may be utilized toward future selection of possible treatment options for migraine.

Aberrant cerebellar Purkinje cell activity as the cause of motor attacks in a mouse model of episodic ataxia type 2.

Many cerebellar-induced neurological disorders, such as ataxias and cerebellar-induced dystonias, are associated with abnormal Purkinje cell activity. In tottering mice, a well-established mouse model of episodic ataxia type 2 (EA2), cerebellar Purkinje cells are required for the initiation of motor attacks. How Purkinje cells contribute to the initiation of attacks is not known, and to date there are no reports on the activity of Purkinje cells during motor attacks in the tottering mice. Here, we show that tottering Purkinje cells exhibit high-frequency burst firing during attacks, reminiscent of other mouse models of cerebellar-induced motor dysfunction. We recorded the activity of Purkinje cells in awake head-restrained tottering mice at baseline, or during caffeine-induced attacks. During motor attacks, firing of Purkinje cells transformed to high-frequency burst firing. Interestingly, the extent to which the activity of Purkinje cells was erratic was correlated with the severity of the motor dysfunction. In support of a causal role for erratic activity in generating motor dysfunction, we found that direct infusion of the small conductance calcium-activated potassium (SK) channel activator NS309 into the cerebellum of tottering mice in the midst of an attack normalized the firing of Purkinje cells and aborted attacks. Conversely, we found that inducing high-frequency burst firing of Purkinje cells in wild-type animals is sufficient to produce severe motor signs. We report that erratic activity of wild-type Purkinje cells results in ataxia and dystonic postures. Moreover, this aberrant activity is the cause of motor attacks in the tottering mice.

Cerebellar involvement in migraine.

Background Although there is a great wealth of knowledge about the neurobiological processes underlying migraine and its accompanying symptoms, the mechanisms by which an attack starts remain elusive, and the disease remains undertreated. Although the vast majority of literature focuses on the involvement of the trigeminovascular systems and higher systems it innervates, such as thalamic and hypothalamic nuclei, several lines of evidence implicate the cerebellum in the pathophysiology of migraine. Aim In this review, we aim to summarize potential cerebellar involvement seen from different perspectives including the results from imaging studies, cerebellar connectivity to migraine-related brain structures, comorbidity with disorders implying cerebellar dysfunction, similarities in triggers precipitating both such disorders, and migraine and cerebellar expression of migraine-related genes and neuropeptides. We aim to inspire an increase in interest for future research on the subject. Conclusion It is hoped that future studies can provide an answer as to how the cerebellum may be involved and whether treatment options specifically targeting the cerebellum could provide alleviation of this disorder.